Abstracts from the 8th International Conference on cGMP Generators, Effectors and Therapeutic Implications

This work was supported by a restricted research grant of Bayer AG

Molecular and functional compartmentation of cGMP in the heart

Cyclic GMP (cGMP) is a second messenger that regulate cardiovascular functions and therapies that increase cGMP shows beneficial effects. However, the mechanisms behind some of these effects in the heart are elusive. In the heart, cGMP is synthesized by either soluble guanylyl cyclase (sGC) or natriuretic peptide-dependent activation of GC-A and GC-B. The aim of the thesis was to investigate subcellular cGMP signalling in cardiomyocytes by measuring cGMP using localized FRET biosensors to unravel the signalling pathways involved in myocardial relaxation and apoptosis. Using biosensors, we show that GC-B increases cGMP around the sarcoplasmatic reticulum and troponin I, thus reducing myocardial contractility and accelerating myocardial relaxation. Furthermore, stimulation of both GC-A and GC-B increase cGMP around the outer mitochondrial membrane, and this protects cardiomyocytes from apoptosis. Since there are few FRET biosensors available for cGMP, we also developed novel biosensors with high affinity for cGMP, and show their use in several cell types. Overall, FRET biosensors are useful tools to reveal organization of signalling pathways and how these mediate functional responses

Phosphodiesterases and Compartmentation of cAMP and cGMP Signaling in Regulation of Cardiac Contractility in Normal and Failing Hearts

Cardiac contractility is regulated by several neural, hormonal, paracrine, and autocrine factors. Amongst these, signaling through β-adrenergic and serotonin receptors generates the second messenger cyclic AMP (cAMP), whereas activation of natriuretic peptide receptors and soluble guanylyl cyclases generates cyclic GMP (cGMP). Both cyclic nucleotides regulate cardiac contractility through several mechanisms. Phosphodiesterases (PDEs) are enzymes that degrade cAMP and cGMP and therefore determine the dynamics of their downstream effects. In addition, the intracellular localization of the different PDEs may contribute to regulation of compartmented signaling of cAMP and cGMP. In this review, we will focus on the role of PDEs in regulating contractility and evaluate changes in heart failure

Compartmentation of cGMP Signaling in Induced Pluripotent Stem Cell Derived Cardiomyocytes during Prolonged Culture

The therapeutic benefit of stimulating the cGMP pathway as a form of treatment to combat heart failure, as well as other fibrotic pathologies, has become well established. However, the development and signal compartmentation of this crucial pathway has so far been overlooked. We studied how the three main cGMP pathways, namely, nitric oxide (NO)-cGMP, natriuretic peptide (NP)-cGMP, and β3-adrenoreceptor (AR)-cGMP, mature over time in culture during cardiomyocyte differentiation from human pluripotent stem cells (hPSC-CMs). After introducing a cGMP sensor for Förster Resonance Energy Transfer (FRET) microscopy, we used selective phosphodiesterase (PDE) inhibition to reveal cGMP signal compartmentation in hPSC-CMs at various times of culture. Methyl-β-cyclodextrin was employed to remove cholesterol and thus to destroy caveolae in these cells, where physical cGMP signaling compartmentalization is known to occur in adult cardiomyocytes. We identified PDE3 as regulator of both the NO-cGMP and NP-cGMP pathway in the early stages of culture. At the late stage, the role of the NO-cGMP pathway diminished, and it was predominantly regulated by PDE1, PDE2, and PDE5. The NP-cGMP pathway shows unrestricted locally and unregulated cGMP signaling. Lastly, we observed that maturation of the β3-AR-cGMP pathway in prolonged cultures of hPSC-CMs depends on the accumulation of caveolae. Overall, this study highlighted the importance of structural development for the necessary compartmentation of the cGMP pathway in maturing hPSC-CMs

FRET-based cyclic GMP biosensors measure low cGMP concentrations in cardiomyocytes and neurons

Several FRET (fluorescence resonance energy transfer)-based biosensors for intracellular detection of cyclic nucleotides have been designed in the past decade. However, few such biosensors are available for cGMP, and even fewer that detect low nanomolar cGMP concentrations. Our aim was to develop a FRET-based cGMP biosensor with high affinity for cGMP as a tool for intracellular signaling studies. We used the carboxyl-terminal cyclic nucleotide binding domain of Plasmodium falciparum cGMP-dependent protein kinase (PKG) flanked by different FRET pairs to generate two cGMP biosensors (Yellow PfPKG and Red PfPKG). Here, we report that these cGMP biosensors display high affinity for cGMP (EC50 of 23 ± 3 nM) and detect cGMP produced through soluble guanylyl cyclase and guanylyl cyclase A in stellate ganglion neurons and guanylyl cyclase B in cardiomyocytes. These biosensors are therefore optimal tools for real-time measurements of low concentrations of cGMP in living cells